PL189514B1 - Method of obtaining tricaronylmetal compounds of fac structure and their application in labelling biologically active substrates - Google Patents

Abstract

1 . A method for preparing a tricarbonyl metal compound with the structure fac of the general formula fac-[M (CO )3(OH2)3]+ (I) in which M is Mn, 9 9 m T c, 18 6 R e or 188 Re, by reacting the metal in in the form of permetalate with carbon monoxide and a reducing agent, characterized in that the mixture of a base, a reducing agent soluble in water but not substantially decomposed by water, and optionally a stabilizing agent is dissolved in water containing a solvent system containing a metal solution in the form of permanganate, pertechnetate or perrhenate in the presence of carbon monoxide 9. Method of preparing a tricarbonylmetal coordination compound with the structure fac with the general formula fac-[M (CO)3(X)2Li]n (II), fac - [M (CO)3(X)L 2] n ( I I I ) or b f a c - [ M ( CO ) 3 L 3 ] n ( I V ) in which: M is M n, 9 9 m Te, 186R e or 188Re; L1 is a monodentate ligand, L2 is selected from the group consisting of a bidentate ligand and two monodentate ligands, and L3 is selected from the group consisting of a tridentate ligand, a monodentate ligand, and a bidentate ligand and three monodentate ligands, X is H2O or halide ion n is the sum of the charges of the ligands L 1 or L 2 or L 3 1 X increased by one charge + ; characterized in that the compound of general formula I obtained in the manner claimed in claim 1 is reacted with the ligand L1 or L2 or L3, optionally in the presence of a halide. 12. Method for preparing a tricarbonyl metal coordination compound with the structure fac having the general formula fac-[M (CO)3(X)2L 1]n (II), fac- M (CO)3(X) L 2]n (III) or fac-[M (CO )3 L 3] n (IV), M is Mn, 9 9 m T c , 186R e or 188Re, L 1 is a monodentate ligand, L 2 is selected from the group consisting of a bidentate ligand 1 two monodentate ligands, and L3 is selected from the group consisting of a tridentate ligand, a monodentate ligand and a bidentate ligand and three monodentate ligands, X is H2O or a halide ion n is the sum of the charges of the ligands L1 or L2 or L3 and one charge +, I know the other one in that .................... . . . PL PL PL

Description

The invention relates to a process for the preparation of fac-structure metal tricarbonyl compounds and further metal tricarbonyl coordination compounds of the fac structure. Furthermore, the invention also relates to a kit for the preparation of labeled compositions and a kit for the preparation of diagnostic or therapeutic pharmaceutical compositions.

The obtained fac-structure metal tricarbonyl compounds can be used for labeling biologically active substrates and other ligands.

The use of metal complexes with many types of radionuclides has become the main diagnostic tool of nuclear medicine and later also of healing. Metal complexes are often combined with biologically active substrates that act as targeting agents.

One of the most widely used methods of metal labeling of biologically active substrates, such as proteins, peptides, sugars and less biologically active compounds, is to stabilize a part of the M (V) = O molecule with the (radioactive) metal from group 7B of the periodic table using various tetrabate ligands. Following reduction, the intermediate stage of the M (V) = O molecule is stabilized with a large amount of an auxiliary ligand such as glycoheptonate, which is then displaced by a chelator linked to the system to be labeled. This method has proved successful in many cases, but has several basic disadvantages such as the required high forcepsiness and size of the ligand and the difficulty of synthesizing and attaching such a ligand.

It is known in the art (Alberto et al., J. Nuc. Biol. And Med. 1994, 38, 388-90) that tricarbonyl metal complexes with the structure of fac radioactive metals from group 7B of the periodic table are very convenient starting materials for substitution reactions. both in organic solvents and in water, because these compounds are stable in water for weeks, even when in contact with air. Therefore, these compounds are very useful for labeling biologically active substrates such as amino acids, peptides, proteins, sugars and all receptor binding molecules. However, the main disadvantage of these compounds so far has been that they can only be obtained from carbonylation reactions taking place at high temperatures and with the aid of a self-igniting and toxic and therefore dangerous reducing agent such as BH3 (Alberto et al., Low CO pressure synthesis of ( NEt) 2 [MX3 (CO) 3] (M = Tc, Re) and its Substitution Behavior in Water and Organic Solvents. Technetium in Chemistry and Nuclear Medicine, No 4, Cortina International, Milano, 1994).

The object of the present invention is to provide a process for the preparation of fac (radioactive) group 7B metal tricarbonyl compounds with readily available and low-toxic starting materials at moderate temperature and normal CO pressure in moderate time and with high yield. Such a method can be very useful for the synthesis of diagnostic and therapeutic agents, especially for the synthesis of diagnostic and therapeutic agents containing radioactive metals with a short average lifetime in order to make these labeled compounds available to modestly equipped hospital laboratories. When the diagnostic agent is labeled with a radionuclide, it can be detected by so-called single photon emission computed tomography (SPECT and SPET), and when labeled with a paramagnetic metal atom then it can be detected by magnetic resonance imaging.

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The above-mentioned object can be achieved, according to the present invention, by using the process for the preparation of a compound of the general formula fac- [M (CO) 3 (OH ₂ ) 3] ⁺ (1) in which M is Mn, ^9m Tc, ¹⁸⁶ Re or ¹⁸⁸ Re , by reacting the metal permetalate (as MO4) with carbon monoxide and a reducing agent, wherein a mixture of a base, a water-soluble but essentially non-degradable reducing agent, and an optional stabilizing agent is dissolved in the water containing the system solvent based solution of the metal in the form of permanganate, pertechnetate or perrhenate in the presence of carbon monoxide.

A preferred metal M is ^9m Tc, 1 Re or 1 ⁸⁸ Re, as these radionuclides, when used in diagnostic or therapeutic agents, have the advantage that they can be used at very low concentrations which minimize the risk of toxicity.

The term "substantially non-degradable by water" means that upon addition of a solution of permanganate, perhenethane, or perrhenate in water, the reaction rate of the decomposition of the reducing agent by water is zero or very slow compared to the rate of reaction of the reducing agent with the permanganate, perthenate or perrhenate, such that the reaction with the above-mentioned permetalate is complete when a sufficient amount of reducing agent is still present.

It was very surprising that the quantitative reduction of the permetalates in the water containing the solvent system could be achieved at moderate temperature and within a reasonable time with reducing agents that are nucleophilic and generally considered less reactive than the prior art electrophilic reducing agent BH3.

The process of the invention can be carried out by simply mixing the permetalate solution with the other reactants in the presence of carbon monoxide. The permetalate solution may optionally contain halide ions needed to elute the permetalate from the generator. Carbon monoxide can be supplied by using a closed system containing the appropriate amount, or by passing carbon monoxide gas through the solution.

The base used is preferably an inorganic base selected from the group of stable hydroxides and carbonates such as NaOH, KOH, NaHCO 3, Na ₂ CO 3, KHCO 3, K ₂ CO 3, Ca (OH) ₂ and Mg (OH) _2. Na2CC> 3 is most preferred. The base is added in a molar ratio to the reducing agent ranging between 0.1 and 2, and preferably about 0.35.

The reaction can be carried out with or without the addition of a stabilizing agent. Gentisan (2,5-dihydroxybenzoate), glycoheptonate, citrate or tartrate can be used as a stabilizing agent. A preferred stabilizing agent is a tartrate, e.g. such as NaK tartrate. The stabilizing agent is added to the reaction mixture in such an amount that its concentration is greater than that of the metal to be reduced.

Several reducing agents may be used for reduction such as a borane anion (BH4 ') or a substituted borane anion in which up to three hydrogen atoms that the borane anion contains are replaced with neutral substituents. Examples of such inert groups are alkoxy or alkylcarbonyloxy groups having 1 to 10 carbon atoms and cyano groups. The counterion of the reducing group may be a metal from group 1A or 2A of the Periodic Table of the Elements or zinc, or an ammonium group or a tetrasubstituted ammonium group or a tetrasubstituted phosphonium ion in which the four substituents each independently may be an alkyl group having 1 to 10 carbon atoms, a hydroxyalkyl group, or an alkoxyalkyl group having 2 to 10 carbon atoms or an aryl group.

A preferred reducing agent is a borane anion, especially in the form of a compound such as sodium borohydride, potassium borohydride, lithium borohydride or zinc borohydride. The most preferred reducing agent is sodium borohydride.

The reducing agent reacts with the permetalate in a molar ratio above 3. The reduction reaction can be carried out at a temperature between 20 ° C - 100 ° C. The preferred reaction temperature is about 75 ° C. The reaction mixture can be heated in the normal manner or with microwave radiation. The reaction can also be performed by using ultrasound, e.g. in an ultrasonic bath at room temperature, achieving the same reaction rate at a lower temperature.

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The resulting compound of formula (I) is very suitable for labeling biologically active substances such as amino acids, peptides, proteins, sugars and small receptor or cell binding molecules.

Examples of peptides that can be labeled are: growth factors, somatostatin, bombesin, insulin, LHRH, gastrin, gastrin-releasing peptide, thyrotropin-releasing hormone, thyroid-stimulating hormone, prolactin, vasoactive intestinal peptide (VlP), pituitary adenylate-activating polypeptide (PACA-P) , angiotensin, neurotensin, interferons, IL-1, IL-4 and IL-6, monoclonal antibodies and analogs and derivatives thereof. These peptides, when labeled with an appropriate labeling substance, can be used, for example, for the detection and localization or treatment of malignant human tumors.

Examples of sugars that can be labeled are glucose and deoxyglycose and derivatives of these compounds.

Small receptor binding molecules are non-peptide molecules which bind to receptors and normally have a molecular weight below about 500 Daltons.

Examples of small receptor binding molecules that can be labeled are substances of the serotonergic system, such as those described in WO 96/30054, or substances of the dopaminergic system (e.g. rocloprid, β-CIT, lisuride), the cholinergic system (e.g. epibatidine ), the glutamatergic system (e.g. mematin) or the benzodiazepine system (e.g. flumazenil, iomazenyl). Examples of metabolically active molecules that can be labeled are: DOPA, tyrosine, mlBG, MAO-I and their analogues.

Examples of cells that can be labeled are red and white blood cells.

Labeling the (biologically active) substrate with the compound of general formula I gives a further coordination compound of the general formula fac- [M (CO) 3 (X) 2L,] ⁿ (II), fac- [M (CO) 3 (X ) L ₂ ] ⁿ (III) or fac- [M (CO) 3L3] ⁿ (IV) where:

M is Mn, ⁹⁹ⁿⁿ Tc, '^ Re or'Re;

Lj is the monodentate ligand,

L2 is selected from the group consisting of a bidentate ligand and two monidentate ligands, and

L3 is selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monodentate ligands;

X is H ₂ O or a halide ion;

n is the sum of the charges of the ligands L 'or L2 or L3 and X increased by one + charge.

After the labeling reaction, ligand X is typically H 2 O. However, one of the H ₂ O ligands may be replaced with a halide ion, if available, to neutralize the charge of the complex. This is often the case with compounds of general formula III.

When the L ', L2 or L3 ligand before and / or after labeling with a fac structure tricarbonyl metal compound is a biologically active molecule, the present invention provides easy access to compounds that can be directly used as diagnostic and therapeutic agents.

Examples of monodentate ligands within the definition of Li, L2 and L3 are (biologically active) substrates bearing groups of compounds such as phosphines, isonitriles, nitriles, imidazoles, thioethers and pyridine-like aromatic amines.

Examples of bidentate ligands within the definition of L2 and L3 are (biologically active) substrates bearing pyridine, imidazole or pyrazole groups such as histidine, histamine, functionalized imidazole systems, bidentate thioethers, bidentate isocyanides, Schiff base ligands and picolinic acid.

Examples of tridentate ligands within the definition of 1.3 are: trispyrazolylborane, trispyrazolylmethane, trisimidazolylborane, trispyrazolylmethane, 1,4,7-trithiacyclononate (9-anS3), 1 triazacyclononane (9-anN3), histidine, cysteine, and a substituted methionine group thioether and cyclopentadienyl derivatives.

In some cases, it may be advantageous to produce radiolabelled bioactive compounds in a single step. According to the present invention, this aim

189 514 can be achieved using a method for the preparation of a compound of the general formula fac- [M (CO) 3 (X) 2Li] n (II), fac- [M (CO) ₃ (X) ₂ L,] ⁿ (II), fac - [M (CO) 3 (X) L ₂ ] ⁿ (III) or fac- [M (CO) ₃ L ₃ ] n (IV) where:

M is Mn, ^99m Tc, ¹⁸⁶ Re or ¹⁸⁸ Re;

Li is a monodentate ligand,

L2 is selected from the group consisting of a bidentate ligand and two monidentate ligands, and

L3 is selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monodentate ligands;

X is H 2 O or a halide ion;

n is the sum of the charges of the Li or L2 ligands or L3 and X increased by one + charge; consisting in that a mixture of the base, ligand Li or L2 or L3 of a water-soluble but essentially not decomposed by water reducing agent and optionally a stabilizing agent are dissolved in water containing a solvent system containing a metal solution in the form of permanganate, perethylene or perrhenate in the presence of carbon monoxide and optionally in the presence of a halide. Particularly in the case of radiolabelled compounds, it is often impossible to provide the user with a ready-to-use composition due to the often short shelf life of the radiolabelled compound and / or the short half-life of the radionuclide used. In this case, the user will perform the metal labeling reaction in a clinical hospital or laboratory. To this end, the various reaction components are then offered to the user in the form of a so-called "kit". It is obvious that the manipulations necessary to accomplish the desired reaction should be as simple as possible to enable the user to produce a radiolabeled composition from the kit using the equipment at his disposal. Therefore, the invention also relates to a kit for making a labeled composition, the labeled composition containing a compound of formula I as the labeled agent.

Such a kit for labeling the biologically active substrates of the present invention comprises (i) a reducing agent that is water-soluble but substantially not degraded by water, (ii) a base, (iii) a stabilizing agent and / or chelator if desired, and (iv) if desired , one or more inert pharmaceutically acceptable carriers and / or molding agents and / or auxiliaries, at least one of components (i) to (iv) is stored in a container having an atmosphere containing an appropriate amount of carbon monoxide, components (i) to (iv) ) optionally independently being combined, and (v) instructions for use with a prescription for reacting the ingredients of the kit with a metal selected from Mn, Tc 9 ^{m 9, l8} or 6RE and ^"8 Re in the form of a permetallate solution.

The advantage of the present invention, which discloses an easy process for the preparation of fac-structure tricarbonyl metal compounds in a time interval sufficiently comparable to the half-life of the radioisotope used, and with high efficiency, is that a kit for labeling biologically active substrates can be produced using these fac-structured metal tricarbonyl compounds.

In some cases, it may be advantageous to include a bioactive substrate in the kit, such that a kit is obtained for the preparation of a radioactive pharmaceutical composition. Alternatively, a biologically active compound is formed by reacting a ligand with a fac-structure metal tricarbonyl compound.

Such a kit for the preparation of diagnostic and therapeutic pharmaceutical compositions, according to various embodiments of the present invention, comprises (i) a suitable substrate to be labeled with a metal selected from the group consisting of Mn, ^m Tc, ° Re or ³⁸⁸ Re, (ii) a soluble reducing agent in water, but not substantially degraded by water, (iii) a base, (iv) if desired, a stabilizing agent and / or chelator, (v) if desired, one or more inert pharmaceutically acceptable carriers and / or molding agents and / or agents auxiliaries, at least one of components (i) to (v)

189 514 is stored in a container having an atmosphere containing an appropriate amount of carbon monoxide, components (i) to (v) optionally independently mixed with each other, and (vi) instructions for use with a recipe for reacting kit components with a metal in the form of a permetalate solution.

The preparation of diagnostic and therapeutic pharmaceutical compositions from the above-mentioned kit containing a (biologically active) substrate can be carried out in two alternative embodiments. According to a first embodiment, first a fac-structure metal tricarbonyl compound is produced and then reacted with the substrate to be labeled. According to a second embodiment, the reduction step is performed in the presence of the substrate to be labeled, leading directly to the labeled compound.

The following examples will illustrate the invention in more detail.

Example 1. Synthesis of [ ^99m Tc (OH2) 3 (CO) 3] ⁺ from aqueous solution

The following chemicals were placed together in a 10 mL resealable vial: 5.5 mg NaBH4, 4 mg KHCO3, and 20.0 mg NaK tartrate. The vial is capped with a serum and flushed for 10 minutes with carbon monoxide gas using a syringe. Then 3 ml of 0.9% NaCl solution from a Mo-99 / Tc-99m generator, having an activity of about 100 mCi, was added through the septum, heated to 75 ° C over 30 minutes, and then cooled to room temperature. The product was analyzed by TLC on standard Merck silica plates with a mixture of methanol / conc. HCl = 99/1 as the mobile phase followed by analysis of the silica plates via a radioactivity scanner. The yield of pertechnetane reduction to [ ^m Tc (OH ₂ ) 3 (CO) 3] ⁺ with the fac structure was> 95% by TlC. After neutralizing the solution with PBS (phosphate buffer (pH = 7.4, saline 0.9%)), a neutral physiological solution was obtained, suitable for labeling.

Table 1 shows that by using different reaction conditions [9 ^9m Tc (OH2) 3 (CO) 3] + solutions having an activity up to 700 mCi can be obtained.

Table 1

Production of [ ^99m Tc (OH2) 3 (CO) ₃ ] ^b under various reaction conditions

No. Let's stabilize the center TcO4 solution volume (ml) Activity (mCi) Solvent Temperature <° 0 Time reaction (min) Yield (TLC) (%) 1 NaK tartrate -) 3 »100 H2O 75 thirty > 95 2 NaK tartrate. 3 »400 H ₂ O 75 thirty > 95 3 3 NaK tartrate 3 3 »700 ** H2O 75 thirty > 95 4 NaK tartrate 3 Well.* H ₂ O 75 thirty > 95 5 NaK tartrate 6 Well.* H2O 75 thirty > 95 6 *** NaK tartrate 3 3 Well. * H2O 75 thirty 40 7 - 3 Well.* H2O 75 thirty 70 8 Na citrate 3 n.o * H2O 75 thirty twenty 9 Na formate 3 no * H2O 75 thirty 35 10 NaK tartrate 3 Well.* H2O / EtOH 80/20 75 thirty > 95 11 NaK tartrate 3 Well.* H2O 100 10 80

* Activities were not accurately marked but ranged between 50 and 200 mCi. ** Activity was determined after dilution to 1% o.

*** 4.0 mg Ca (OH) 2 was used as base instead of 4.0 mg

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Example 2. Synthesis of the complex composition [ ⁹⁹ⁱⁿ TcL ₃ (CO) ₃ ]

2.1. Synthesis of [ ^m Tc (his) (CO) a] via [99 ^m Tc (OH2b (CO) ₃ ] ⁺ .

After completion of the reaction described in Example 1, 0.1 pmol of histidine was added to the solution at pH 7.4. According to TLC, the reaction was complete after 1 hour.

When 0.01 pmol of histidine was added at room temperature, the reaction took 5-10 hours to complete; at 70 ° C the reaction was complete in less than 1 hour.

2.2. Direct synthesis of [mTc (his) (CO) ₃ ],

The experiment was performed as described in Example 1. Simultaneously with adding the generator eluate to the cold vial, 0.03 pmol of histidine was added to the cold vial. After heating for 30 minutes, [mTc (his) (CO) ₃ ] ^{+ was} obtained in almost quantitative yield by TLC.

2.3. Synthesis of [ ⁹⁹ mTc ((lys-gly- (his) ₅ ) (CO) 3] + via [mTc (OH ₂ ) 3 (CO) 3] ⁺ .

After completion of the reaction described in Example 1, 500 pmol of the octapeptide lys-gly-gly- (his) ₃ was added to the solution. According to TLC, the reaction was complete after 1 hour at room temperature.

When 300 pmol of lys-gly-gly- (his) was added, the reaction was complete after 3 hours.

2.4. Brief description of the preparation of further complexes of the composition [mTcL (CO) ₃ ]

[99mTc (OH2) ₃ (CO) ₃ ] ^{+ is} prepared as described in Example 1 and neutralized. The ligand (see Fig. 1) is added using the complexation time, temperature, amount and concentration given in Table 2. Table 2 also shows the yield obtained as determined by the TLC analysis described in Example 1 and the possibility of carrying out the one-pot reaction.

Table 2

Production of [ ⁹⁹ 'TcL (CO) 3] with different ligands and reaction conditions

Ligand (fig. 1) pH. time temp. concentration total quantity performance One vessel **** 1 PBS thirty' 70 ° C 3 pM 5 nmol 92% partly 2 PBS 2-3 h 37 ° C 20 pM 10 nmol 74% Yes 3 PBS 4 h 37 ° C 100 pM 50 nmol 89% Yes 4 PBS 6 h 37 ° C 100 pM 50 nmol 72% - 5 PBS / CH30H 1 h 50 ° C 100 pM 100 nmol 65% - 6 PBS 1 h + 1 h 50 ° C * 97% + 30% - 7 *** PBS thirty' 75 ° C 25 pM 25 nmol 95% Yes 7 *** PBS 15 ' ** 25 pM 25 nmol 95% Yes 8 PBS 1 h 75 ° C 10 pM 2 nmol 95% Yes

* 50 pl 10 3m ligand, then 50 pl D ^ M glucose ** Ultrasound treatment for 15 minutes at room temperature *** After reaction, the product stored for 23 hours at 37 ° C proved to be stable **** Possibility of synthesis in one pot

H-NMR spectrum was prepared from the compound obtained from ligand 5 (structure - see below) by complexation with "cold" [Te (OH ₂ ) 3 (CO) 3] +. A: aromatic region of NMR spectrum before complexation: H-NMR _(CDCl3) [tp, δ in ppm] = 8.75 (1H, d) (a), 8.36 (II-I, s) (e) 8.14 (IH, d) (d), 7.99 (IH, t) (b), 7.55 (IH, m) (c), 6.07 (IH, s) (f). B: aromatic region of NMR spectrum after complexation: Ή-NMR (CDCl ₃ ) [tp, δ in ppm] - 9.12 (1H, d) (a), 8.51 (1H, s) (e), 8, 37 (IH, t) (b), 8.19 (IH, d) (d), 7.87 (IH, m) (c), 6237 (IH, s) (f).

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Example 3. Labeling of antibodies with [mTc (OH2) 3 (CO) 3] ⁺

3.1. Marking as a function of concentration

The labeling kinetics were tested as a function of the Mab concentration. A concentration above 2-3 mg / ml gives a quantitative labeling after 2 hours, while below 1 mg / ml the yield by TLC is 40-50%.

3.2. In vitro biological activity of the labeled material monoclonal antibody 35 (Mab-35)

The amount of [mTc (OH2) (CO) 3] ⁺ obtained in Example 1 was used to label 1.2 mg of Mab-35. After 3 hours of incubation at 37 ° C, the Mabs were separated by means of a PD-10 size exclusion gel column chromatography with a yield of 38%. The labeled Mab when tested by Lindmo (T. Lindmo, PA Brunn, Methods in Enzymology 1986, 121, 678) showed 100% biological activity.

Example 4. Labeling of His-Neurotensin (8-13) by [ ^99rn Tc (OH2) 3 (CO) 3l ⁺

0.9 ml of [99m Tc (OH2) 3 (CO) 3] ⁺ obtained in Example 1 was mixed with 0.1 ml of 10 ^'3 M HisNeurotensyny ^ -13) (HRRPYIIL) and kept in a sealed tube at 75 ° C for one hour. At the end of this time, the reaction mixture was cooled to room temperature. The yield, as determined by a reverse-phase HPLC column, is> 95%. The Kd of this compound on HT29 colon cancer cells was 1.0 nM.

Example 5. Labeling of the recombinant proteins of scFv fragment with 6 x His-tag by [99mτc (CH2) 3 (CC) _3] ^ł

0.1 ml [ ^IU Tc (OH2) 3 (CO) 3] obtained in example 1 was mixed with 0.1 ml of IM MES pH 6.2 buffer and 0.1 ml of 150 pM 20 scFv-6 x His and kept at temperature 37 ° C for 20 min. At the end of this time, the reaction mixture was run on a Sephadex® G-25 Superfine column. Typical inclusions of mTc are 70% to 84%, with biological activities (measured using the Lindmo assay mentioned in Example 3) ranging from 57% to 90%. The Kd values were not significantly affected by the inclusion of Te done using BIAcore measurements showed the unlabeled scFv: 0.5 x 10 ⁸ M, by ^ Tc labeled scFv: 1 x 10 M, scFv labeled with 1: 4 x 10 ^-8 M .

Example 6. Labeling of biotin by [99mTc (OH2) 3 (CO) 3] +

[99mTc (OH2) 3 (CO) 3] ⁺ prepared as described in Example 1. 1300 µL of a 10 µM solution of biotin hydrazide in pyridine was added to 2 ml of the carbonyl-Tc compound and incubated at 50 ° C for 2 hours to give the compound labeled by Te with a purity of 50%. This compound was purified by loading it into a balanced (5 ml Me-OH / H ₂ O = 1/1) SepPac column and eluting the by-products with 2 ml of water followed by 4 ml of Me OH / H 2 O = 1/1, then eluting the desired product with 2 ml of MeOH. Final purity of the compound was 98%. Stability control: no degradation in methanol after 24 hours; 32% degradation in PBS buffer after 24 hours. Streptavidin-beads binding test results: 0.5% non-specific binding and 81% specific binding.

Claims (20)

1 A method for producing a tricarbonyl metal compound of the fac structure of the general formula fac- [M (CO) ₃ (OH ₂ ) ₃ ] ⁺ (I) where O is On, "Tc, ^IS6 Re or ^I88 Re, by reacting the metal in the form of a permetalate with carbon monoxide and a reducing agent, characterized in that the mixture of the base, the reducing agent is water-soluble but not substantially decomposed by water, and optionally the stabilizing agent is dissolved in water containing a solvent system containing a metal solution in the form of permanganate, perethylene or perrhenate in the presence of carbon monoxide 9. The method of producing a fac-tricarbonyl coordination compound with the general formula fac- [M) COI) (; XI ^^ i] ⁿ ) II), fac- M (CO) ₃ (X) L ₂ ] ⁿ (III) or fac- [M (CO) jLj] (IV) where: "", O means He, "Te, i ⁸⁶ Re or i" ^s Re; Li is a monodentate ligand, L (is selected from the group consisting of a bidentate ligand and two monodentate ligands, and L) is selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monodentate ligands, X is H (O or the halide ion n is the sum of the charges of the Li or L ligands (or L ₃ 1 X increased by one + charge;. characterized in that the compound of general formula I obtained as claimed in claim 1 1 is reacted with a ligand Li or L (or L), optionally in the presence of a halide. 12. The method of producing a tricarbonyl metal coordination compound with the structure fac of the general formula fac-iOmOórabLi]) II), fac- O) C0l)) xiL (] n (Ili) or fac-McOjUr (IV), O means On, Tc, i ⁶ Re or i ' ⁸ Re, Li is a monodentate ligand, L (is selected from the group consisting of a bidentate ligand and two monidentate ligands, and L) a test selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monodentate ligands, X is H (O or the halide ion n is the sum of the charges of the ligands Li or L (or L) 1 X increased by one + charge, characterized in that ......... 189 514 The method for the production of a metal tricarbonyl compound with the fac structure of the fac coordination tricarbonyl metal compounds with the fac structure and a kit for the production of character compositions and a kit for the production of diagnostic or therapeutic pharmaceutical compositions Patent claims 1. A method for the production of a tricarbonyl metal compound of the fac structure of the general formula fac- [M (CO) 3 (OH ₂ ) ₃ ] ⁺ (I) in which M is Mn, ^99m Tc, ¹⁸⁶ Re or 1 ⁸⁸ Re, by reaction of the metal in permetalate form with carbon monoxide and a reducing agent, characterized in that the mixture of the base, the reducing agent soluble in water but not substantially decomposed by water, and optionally the stabilizing agent is dissolved in the water containing the solvent system containing the metal solution in the form of permanganate, penthenate or perrhenate in the presence of carbon monoxide. 2. The method according to p. The process of claim 1, wherein the base is an inorganic base, preferably selected from the group consisting of NaOH, KOH, NaHCO3, Na ₂ CO ₃ , KHCO3, K2CO3, Ca (OH) 2 and Mg (OH) 2. 3. The method according to p. The process of claim 1, wherein the molar ratio of base to reducing agent is between 0.1 and 2, and is preferably about 0.35. 4. The method according to p. The process of claim 1, wherein the reducing agent is selected from the group consisting of a borane anion and a substituted borane anion, in which up to three hydrogen atoms that the borane anion contains are replaced with neutral substituents. 5. The method according to p. The process of claim 4, wherein the reducing agent is a borane anion derived from a salt selected from the group consisting of sodium borohydride, potassium borohydride, lithium borohydride and zinc borohydride. 6. The method according to p. The process of claim 5, wherein the reducing agent is sodium borohydride. 7. The method according to p. 6. The process of claim 6, wherein the molar ratio of reducing agent to permetalate is at least 3. 8. The method according to p. The process of claim 1, wherein the reaction temperature is between 20 ° C and 100 ° C, and is preferably about 75 ° C. 9. The method of producing a fac-tricarbonyl coordination compound with the general formula fac- [M (CO) 3 (X) 2L ₁ ] ⁿ (II), fac- [M (CO) 3 (X) L ₂ ] n (III) or fac- [M (CO) 3L ₃ ] n (IV) where: M is Mn, ^99m Te, R ^ or R ^; L1 is a monidentate ligand, L2 is selected from the group consisting of a bidentate ligand and two monidentate ligands, and L3 is selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monidentate ligands; X is H ₂ O or a halide ion; n is the sum of the charges of the L1 or L2 or L3 and X ligands increased by one + charge; characterized in that the compound of general formula I obtained as claimed in claim 1 1 is reacted with the ligand L1 or L2 or L3, optionally in the presence of a halide. 189 514 10. The method according to p. The method of claim 9, wherein the Li-L3 ligands are biologically active substrates selected from the group consisting of amino acids, peptides, proteins, sugars, small receptor binding molecules and body cells, or derivatives thereof. 11. The marking method according to claim 1 The process of claim 10, wherein the substrate is an amino acid, peptide or protein. 12. The method of producing a fac-tricarbonyl coordination compound of the general formula fac- [M (CO) ₃ (X) 2L,] ⁿ (II), fac- [M (CQ) 3 (X) L2] ⁿ (III) or fac- [M (CO) 3L3] n (IV) where: M is Mn, ^m Tc, ¹⁸⁶ Re or 1 ⁸⁸ Re; L1 is a monidentate ligand, L2 is selected from the group consisting of a bidentate ligand and two monidentate ligands, and L3 is selected from the group consisting of a tridentate ligand, a monidentate ligand and a bidentate ligand, and three monidentate ligands; X is H 2 O or a halide ion; n is the sum of the charges of the L1 or L2 or L3 and X ligands increased by one + charge; characterized in that the mixture of the base, the ligand L1 or L2 or L3, the reducing agent soluble in water but not substantially decomposed by water, and optionally the stabilizing agent is dissolved in the water containing the solvent system containing the metal solution in the form of permanganate, penthenate or perrhenate in the presence of carbon monoxide and optionally in the presence of a halide. 13. The method according to p. The method of claim 12, wherein the L1-L3 ligands are biologically active substrates selected from the group consisting of amino acids, peptides, proteins, sugars, small receptor binding molecules and body cells, or derivatives thereof. 14. The marking method according to claim 1 The process of claim 13, wherein the substrate is an amino acid, peptide or protein. 15. The method according to p. The process of claim 12, wherein the base is an inorganic base, preferably selected from the group consisting of NaOH, KOH, NaHCO3, Na2CO3, KHCO3, K2CO3, Ca (QH) 2 and Mg (OH) 2. 16. The method according to p. The process as claimed in claim 12, characterized in that the molar ratio of base to reducing agent is between 0.1 and 2, and is preferably about 0.35. 17. The method according to p. The process of claim 12, wherein the reducing agent is selected from the group consisting of a borane anion and a substituted borane anion, in which up to three hydrogen atoms that the borane anion contains are replaced with neutral substituents. 18. The method according to p. The process of claim 12, wherein the reaction temperature is between 20 ° C and 100 ° C, and is preferably about 75 ° C. 19. A kit for the preparation of labeled compositions comprising (i) a reducing agent soluble in water but substantially not decomposed by water, (ii) a base, (iii) a stabilizing agent and / or a chelator, if desired, and (iv) one or more if desired. more inert pharmaceutically acceptable carriers and / or molding agents and / or auxiliaries, at least one of components (i) to (iv) is stored in a container having an atmosphere containing a suitable amount of carbon monoxide, components (i) to (iv) optionally independently are mixed together, and (v) the instructions for use with the recipe for reacting the kit components with a metal selected from the group consisting of Mn, ^m Tc, ^Sb Re or ^{, re} Re in the form of a permetalate solution. 20. A kit for the preparation of diagnostic or therapeutic pharmaceutical compositions comprising (i) a suitable substrate to be labeled with a metal selected from the group consisting of Mn, ^m Tc, R Re or R Re, (ii) a reducing agent soluble in water but essentially not decomposed by water, (iii) base, (iv) if desired, mid4 A stabilizer and / or chelator, (v) if desired, one or more inert pharmaceutically acceptable carriers and / or molding agents and / or auxiliaries, at least one of components (i) to (v) is stored in a container having an atmosphere containing a suitable amount of carbon monoxide, components (i) to (v) optionally independently mixed with each other, and (vi) instructions for use with a recipe for reacting kit components with a metal in the form of a permetalate solution.